Cofactors are non-protein chemical compounds that are required by a protein, most commonly an enzyme, to perform a biological activity. Accordingly, man-made systems that are intended to replicate certain biological functions, such as enzymatic activity, require the presence of these cofactors in a way in which they are made available to the proteins in the system in a controlled manner. A common way to control the manner in which a biological, such as a cofactor, is presented and used in a system is by immobilizing the biological to a substrate. This enables the system to guarantee presence of the biological and to control both the location and concentration (if desired) of the biological. Some methods of immobilization can even go so far as to control the position or orientation of a given biological.
However, immobilization of particular biologicals can present problems. First, some systems may require a particular type of substrate or require a substrate formed from a particular type of material that is not necessarily friendly to certain traditional attachment mechanisms. For example, graphene-like materials, which are often used or required in energy-production systems such as fuel cells, do not easily avail themselves to the use of attachment chemistries that rely on the presence of covalent bonds. Moreover, the biological itself may present certain challenges. For example, dynamic cofactors interact with multiple proteins in sequence and thus may require a certain degree of freedom in order to operate.
An exemplary dynamic cofactor is nicotinamine adenine dinucleotide (phosphate) (NAD+(P)/NAD(P)H). The structure of NAD+/NADH (without phosphate group) is shown in FIG. 1. NAD+/NADH is found in all living cells and serves two important functions. First, it is involved in redox reactions, enabling metabolism by trading electrons with other molecules as it alternates between its NAD and NADH forms. Second, it is used in many cellular processes including, acting as a substrate of enzymes that add or remove chemical groups from proteins in posttranslational modifications. Due to these important functions, is an important and highly interesting compound for both research and commercial applications.
Many of these applications, such as, for example, biofuel applications in which anodic enzymes require the presence of NAD(P)+/NAD(P)H as a cofactor for catalysis, would benefit from the ability to immobilize or tether NAD(P)+/NAD(P)H to a substrate, and in particular, the ability to tether NAD(P)+/NAD(P)H to graphene-like structures such as multi-walled carbon nanotubes, single walled carbon nanotubes, graphene, rGO, and other graphene-based or graphene-containing substrates. However, the tethering of NAD(P)+/NAD(P)H has heretofore proven to be difficult, as NAD(P)+/NAD(P)H requires a certain degree of freedom of movement in order to function and thus previous methods that relied on simply immobilizing NAD(P)+/NAD(P)H directly to the surface of the substrate proved to be either of limited value or entirely unworkable.
Accordingly, there is a need for methodologies and chemistries for linking or tethering cofactors such as, but not necessarily limited to NAD(P)+/NAD(P)H, to a substrate, and particularly a graphene-like substrate, that enable suitable immobilization of the cofactors without unduly inhibiting their ability to perform their desired biological function.